Steps involved in the transport of Na+ by the Na,K-ATPase

CENTENO, MERCEDES; FERREIRA GOMES, MARIELA; ROSSI J. P. F. C; MONTES, MÓNICA R.; ROSSI ROLANDO C

Tucuman

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS). IX IberoAmerican Congress of Biop-hysics XLV Reunion Anual SAB 2016; 2016

Sociedad Argentina de Biofísica

The Na,K-ATPase is a membrane-bound ion pump that generates electrochemical gradients for Na+ and K+ across plasma membranes of animal cells. The enzyme oscillates between two major conformations, E1 and E2. Under physiological conditions, E1 binds Na+, ATP and Mg2+ and forms the phosphorylated state E1P with occluded Na+, with release of ADP. After a conformational transition to E2P, Na+ is released and K+ binds and becomes occluded. The subsequent release of K+ leads to E1 and the cycle begins again.In this work we have studied the effects of oligomycin, an antibiotic from Streptomyces diastatochromogenes and epigallocatechin-3-gallate(EGCg), a polyphenolic compound obtained from green tea, as inhibitory agents to isolate the intermediates involved in the transport of Na+. It is proposed that, like oligomycin, EGCc inhibits the enzyme activity by stabilizing the E1 intermediates, thus blocking the E1PE2P conformational change. The advantage of using EGCg rather than oligomycin is its solubility in aqueous solutions (5mg/ml against 25uM/ml). With this aim we measured the ATPase activity, phosphoenzyme level, and the binding/occlusion of Na+ in the enzyme. Results show that EGCg inhibited the ATPase activity with a K0.5 of 1uM as previously reported, the phosphoenzyme level (measured using [γ-32P]ATP) remaining after the addition of K+ was higher in the presence of EGCg than in it´s absence and that in the presence of oligomycin and EGCg the amount of tightly-bound 22Na+ was 18 times higher than with the enzyme thermally inactivated, and the amount of 22Na+ in the enzyme without inhibitors was 8 times higher than with the inactivated enzyme.These results suggest that, like oligomycin, EGCc stabilizes the E1 conformation of the Na,K-ATPase and allows to isolate the intermediates containing occluded Na+.Referencias:Sachs JR (1980) J Physiol, 302: 219-240Ochiai et al. (2009) Biochemical Pharmacology, 78: 1069?1074Agradecimientos:With grants from ANPCYT, CONICET and UBACYT